Throughout this application various publications are referred to in brackets. Full citations for these references may be found at the end of the specification. The disclosures of these publications are hereby incorporated by reference in their entirety into the subject application to more fully describe the art to which the subject invention pertains.
Bacillus anthracis is a gram-positive, facultatively anaerobic, rod-shaped bacterium that is the causative agent of anthrax. It secretes two toxins which are composed of three proteins: protective antigen (PA), edema factor (EF) and lethal factor (LF). The anthrax toxins, like other binary toxins, have distinct subunits involved in the different steps of the toxin's action. The B subunit (PA) is involved in receptor binding and cellular internalization into the cytoplasm, whereas the A subunit (EF and/or LF) bears the enzymatic activity [1]. Anthrax can occur naturally in animals by spore transmission via ingestion, inhalation, or an open skin wound, but it can also be a result of bioterrorism and biological warfare [2].
PA is the component of the currently licensed anthrax vaccine (AVA) that elicits protective antibodies. Recent studies have demonstrated that a strong humoral response to truncated recombinant PA contributes to a protective immune response to anthrax [3, 4]. Accordingly, there is considerable interest in ascertaining the location and immunogenicity of B-cell epitopes on the PA molecule. The development of numerous monoclonal antibodies (mAb) to different epitopes on the PA molecule that influence PA functions, in conjunction with epitope mapping, have provided an opportunity to study the fine antigenic structure of PA. Most of these epitopes have been defined in mice [5-8], in macaques [9], in rabbits [10], as well as in vaccinated humans [11]. Consequently, the epitopes described thus far are localized to three discrete regions within PA. These regions correspond to the 2β2-2β3 loop of domain 2, the domain 3-domain 4 boundary and the small loop of domain 4. The 2β2-2β3 loop of domain 2 is responsible for mediating membrane insertion and many neutralizing murine mAbs target this region [5, 8]. The boundary between domains 3 and 4, which does not have a known functional activity, has been suggested as a region recognized by polyclonal antibodies from vaccinated humans and rabbits [6, 12]. The cellular receptor binding region is localized to the small loop of domain 4, and this region has been described to be recognized by two neutralizing mAbs [7, 9]. With the exception of a neutralizing mAb that bound to PA20 [13], no B-cell epitopes have been reported in domain 1. Therefore, identification of further dominant antigenic epitopes is pivotal for understanding the minimal immunogenic region of PA that will allow for precise direction of potent immune responses.
There is an urgent need for a more effective prophylactic vaccine against anthrax. The treatment of anthrax remains unsatisfactory because of high morbidity and mortality [2] and there are significant drawbacks to the currently licensed vaccine. Consequently, there is considerable interest in the development of passive immune therapies and more effective vaccines. Additionally, anthrax toxin has the ability to impair innate and adaptive immune responses, entering the cytosol of every cell type and altering their signaling pathways, which in turn inhibits the clearance of the bacterium [23]. The present invention answers this need.